Steroid hormone biosynthesis involves several members of the cytochrome P450 superfamily, both mitochondrial and microsomal forms. The long term goal of GM37942 continues to be elucidation of the structure/function relationships of these key enzymes involved in important biological processes. This competitive renewal focuses primarily on two steroid hydroxylases, the mitochondrial cholesterol side chain cleavage cytochrome P450 (P450scc) and the microsomal 17alpha-hydroxylase cytochrome P450 (P45Oc17), utilizing recombinant enzymes produced in E. coli. The Specific Aims are based on published and unpublished results obtained during the present granting period. 1) Utilizing E. coli flavodoxin as a model for the FMN-containing, P450-interacting domain of P450 reductase, amino acid residues on bovine P450c17 required for P450 reductase interaction will be identified by chemical modification and site directed mutagenesis. 2) Random chimeragenesis will be used in E. coli to generate chimeras between different P450s which share common substrates but catalyze reactions at different sites: bovine P450scc/human P450c27 (cholesterol); bovine P450c17/rat P450c17 (progesterone); bovine P450c17/bovine P450c21 (progesterone); bovine P450scc/rat P450c7 (cholesterol). From these chimeras regions required for enzymatic specificity will be identified, which can be analyzed more carefully by site directed mutagenesis. 3) The tertiary structure of bovine adrenodoxin will be determined from existing crystals, while that of P450scc and the adrenodoxin-P450scc complex will be determined once crystals are obtained. Preliminary studies on P450scc have led to a promising starting point for crystallization. Similar strategies will be applied to crystallization of both P450c17 and the P450c17-flavodoxin complex. 4) Interaction of P450c17 with accessory proteins, cytochrome b5 and 3-hydroxysteroid dehydrogenase, will be investigated. Cyt. b5 alters human and bovine P450c17 activities and 3Beta-HSD was purified in association with P450c17 and P450c21 suggesting existence of a complex in endoplasmic reticulum which has physiological relevance. In addition to providing detailed information on P450scc and P450c17, these studies will provide general insight into structure/function relationships of mitochondrial and microsomal P450s. Because P450scc and P450c17 play important roles in regulation of blood pressure, reproduction, growth and stress; these studies will provide new biochemical insights on these essential biological processes.